Transt1 e coli

Multiple sgRNA expression cassettes mixtures (1−2 µg) in an equimolar ratio were digested in a 50-μl reaction with 1 µl of Bsa I-HF (NEB, USA). Digested products were purified using a PCR Pure kit (Magen, China).
Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 5 U of T4 DNA ligase (Thermo, USA), 50 ng of Cas9VL, and 20 molar ratios of each digested sgRNA expression cassette (mixture). Reactions were incubated at 22 °C for 1 h. For two to four sgRNA expression cassettes, 10 µl of the ligation mixture was transformed into 50 µl of chemically competent transT1 E. coli (Transgene, China). In cases with five or more sgRNA expression cassettes, 100 µl of chemically competent transT1 E. coli are required. A schematic diagram of the assembly of multiple sgRNA expression cassettes is shown in Fig. 2d.
We prepared eight pairs of Bsa I-site primers; therefore, eight different sgRNA expression cassettes can be assembled by one-step Golden Gate assembly that is compatible with the BioBrick Standard Assembly. Biobricks of four sgRNA expression cassettes can be amplified using Bb-F and Bb-R primers from four ligation products of sgRNA expression cassettes and assembled into Biobricks site of pHNCas9. This strategy is suitable for ligating more than eight sgRNA expression cassettes into pHNCas9. Bb-F and Bb-R primer are provided in Table S2.

PEASY–AtMYB75 was digested with Kpn I and Spe I restriction enzyme (Thermo, USA). BioBrick OEAtMYB75 fragments were separated by agarose gel electrophoresis and purified using gel Pure kit (Magen, China). pHNCas9 was digested by Kpn I and Xba I restriction enzyme (Thermo, USA) using PCR pure kit. Ligation reactions (10 μl) were set up with 1 × T4 DNA ligase buffer, 1 U of T4 DNA ligase (Thermo, USA), 50 ng of pHNCas9, and five molar ratios of BioBrick OEAtMYB75. Reactions were incubated at 22 °C for 1 h. A total of 5 µl of the ligation mixture was transformed into 25 µl of chemically competent transT1 E. coli (Transgene, China). The new CRISPR vector containing BioBrick OEAtMYB75 was named pHNCas9:OEAtMYB75 (Fig. 7a).